C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy.

Oncolytic adenoviruses, such as Delta-24-RGD (Δ24RGD), are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, Δ24RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with Δ24RGDundergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/2 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with Δ24RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered Δ24RGD-treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with Δ24RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between Δ24RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates Δ24RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.

Plasticity of glomeruli and olfactory-mediated behavior in zebrafish following detergent lesioning of the olfactory epithelium.

The zebrafish olfactory system is a valuable model for examining neural regeneration after damage due to the remarkable plasticity of this sensory system and of fish species. We applied detergent to the olfactory organ and examined the effects on both morphology and function of the olfactory system in adult zebrafish. Olfactory organs were treated once with Triton X-100 unilaterally to study glomerular innervation patterns or bilaterally to study odor detection. Fish were allowed to recover for 4-10 days and were compared to untreated control fish. Axonal projections were analyzed using whole mount immunocytochemistry with anti-keyhole limpet hemocyanin, a marker of olfactory axons in teleosts. Chemical lesioning of the olfactory organ with a single dose of Triton X-100 had profound effects on glomerular distribution in the olfactory bulb at 4 days after treatment, with the most significant effects in the medial region of the bulb. Glomeruli had returned by 7 days post-treatment. Analysis of the ability of the fish to detect cocktails of amino acids or bile salts consisted of counting the number of turns the fish made before and after odorant delivery. Control fish turned more after exposure to both odorants. Fish tested 4 and 7 days after chemical lesioning made more turns in response to amino acids but did not respond to bile salts. At 10 days post-lesion, these fish had regained the ability to detect bile salts. Thus, the changes seen in bulbar innervation patterns correlated to odorant-mediated behavior. We show that the adult zebrafish brain has the capacity to recover rapidly from detergent damage of the olfactory epithelium, with both glomerular distribution and odorant-mediated behavior returning in 10 days.

Observation of eta' decays to pi+pi-pi0 and pi+pi-e+e-.

Using psi(2S)-->pi;{+}pi;{-}J/psi, J/psi-->gammaeta;{'} events acquired with the CLEO-c detector at the CESR e;{+}e;{-} collider, we make the first observations of the decays eta;{'}-->pi;{+}pi;{-}pi;{0} and eta;{'}-->pi;{+}pi;{-}e;{+}e;{-}, measuring absolute branching fractions (37_{-9};{+11}+/-4)x10;{-4} and (25_{-9};{+12}+/-5)x10;{-4}, respectively. For eta;{'}-->pi;{+}pi;{-}pi;{0}, this result probes the mechanism of isospin violation and the roles of pi;{0}/eta/eta;{'}-mixing and final state rescattering in strong decays. We also set upper limits on branching fractions for eta;{'} decays to pi;{+}pi;{-}micro;{+}micro;{-}, 2(pi;{+}pi;{-}), pi;{+}pi;{-}2pi;{0}, 2(pi;{+}pi;{-})pi;{0}, 3(pi;{+}pi;{-}), and invisible final states.

Igf1r+/CD34+ immature ICC are putative adult progenitor cells, identified ultrastructurally as fibroblast-like ICC in Ws/Ws rat colon.

The colon of Ws/Ws mutant rats shows impairment of pacemaker activity and altered inhibitory neurotransmission. The present study set out to find structural correlates to these findings to resolve mechanisms. In the colon of Ws/Ws rats, interstitial cells of Cajal associated with Auerbach's plexus (ICC-AP) were significantly decreased and ICC located at the submuscular plexus and intramuscular ICC were rarely observed based on immunohistochemistry and electron microscopy. Ultrastructural investigations revealed that there was no overall loss of all types of interstitial cells combined. Where loss of ICC was observed, a marked increase in fibroblast-like ICC (FL-ICC) was found at the level of AP. Immunoelectron microscopy proved FL-ICC to be c-Kit(-) but gap junction coupled to each other and to c-Kit(+) ICC; they were associated with enteric nerves and occupied space normally occupied by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r(+)/CD34(+) cells were found in Ws/Ws compared to wild-type rat colons. These CD34(+)/Igf1r(+) cells in the Ws/Ws colon occupied the same space as FL-ICC. Hence we propose that a subset of immature ICC (FL-ICC) consists of adult progenitor cells. Immunohistochemistry revealed a reduction of neurons positive for neuronal nitric oxide synthase. The functional capabilities of the immature ICC and the regenerative capabilities of the adult progenitor cells need further study. The morphological features described here show that the loss of pacemaker activity is not associated with failure to develop a network of interstitial cells around AP but a failure to develop this network into fully functional pacemaker cells. The reduction in nitrergic innervation associated with the Ws mutation may be the result of a reduction in nitrergic neurons.

Determination of the strong phase in D0-->K+pi- using quantum-correlated measurements.

We exploit the quantum coherence between pair-produced D0 and D[over]0 in psi(3770) decays to study charm mixing, which is characterized by the parameters x and y, and to make a first determination of the relative strong phase delta between D0-->K+pi- and D[over]0-->K+pi-. Using 281 pb(-1) of e+e- collision data collected with the CLEO-c detector at Ecm=3.77 GeV, as well as branching fraction input and time-integrated measurements of RM identical with (x2 + y2)/2 and RWS identical with Gamma(D0-->K+pi-)/Gamma(D[over]0-->K+pi-) from other experiments, we find cosdelta=1.03(-0.17)(+0.31)+/-0.06, where the uncertainties are statistical and systematic, respectively. By further including other mixing parameter measurements, we obtain an alternate measurement of cosdelta=1.10+/-0.35+/-0.07, as well as x sindelta=(4.4(-1.8)(+2.7)+/-2.9)x10(-3) and delta=(22(-12-11)(+11+9)) degrees .

Absolute measurement of hadronic branching fractions of the Ds+ meson.

The branching fractions of D(s)(+/-) meson decays serve to normalize many measurements of processes involving charm quarks. Using 298 pb(-1) of e(+)e(-) collisions recorded at a center of mass energy of 4.17 GeV, we determine absolute branching fractions for eight D(s)(+/-) decays with a double tag technique. In particular we determine the branching fraction B(D(s)(+)-->K(-)K(+}pi(+))=(5.50+/-0.23+/-0.16)%, where the uncertainties are statistical and systematic, respectively. We also provide partial branching fractions for kinematic subsets of the K(-)K(+)pi(+) decay mode.

First observation of the decay Ds+-->pn.

Using e+e--->Ds*-Ds+ data collected near the peak Ds production energy, Ecm=4170 MeV, with the CLEO-c detector, we present the first observation of the decay Ds+-->pn. We measure a branching fraction B(Ds+-->pn)=(1.30+/-0.36(-0.16)+0.12)x10(-3). This is the first observation of a charmed meson decaying into a baryon-antibaryon final state.

Study of exclusive charmless semileptonic B decays and |Vub|.

We study semileptonic B decay to the exclusive charmless states pi, rho/omega, eta, and eta;{'} using the 16 fb(-1) CLEO Upsilon(4S) data sample. We find B(B0-->pi-l+nu)=(1.37+/-0.15stat+/-0.11sys)x10(-4) and B(B0-->rho-l+nu)=(2.93+/-0.37stat+/-0.37sys)x10(-4) and find evidence for B+-->eta'l+nu, with B(B+-->eta'l+nu)=(2.66+/-0.80stat+/-0.56sys)x10(-4). From our B-->pilnu rate for q2>16 GeV2 and lattice QCD, we find |Vub|=(3.6+/0.4stat+/0.2syst-0.4thy+0.6)x10(-3) [corrected]

Precision determination of the D0 mass.

A precision measurement of the D0 meson mass has been made using approximately 281 pb(-1) of e+e- annihilation data taken with the CLEO-c detector at the psi(3770) resonance. The exclusive decay D0-->K_{S}phi has been used to obtain M(D0)=1864.847+/-0.150(stat)+/-0.095(syst) MeV. This corresponds to M(D0D*0)=3871.81+/-0.36 MeV, and leads to a well-constrained determination of the binding energy of the proposed D0D*0 molecule X(3872), as Eb=0.6+/-0.6 MeV.

Low doses of ionizing radiation can prevent radiation-induced colonic epithelial hyporesponsiveness to muscarinic agonists.

PURPOSE: Colonic epithelium hyporesponsiveness to different secretagogues occurs after exposure to ionizing radiation, increasing susceptibility to bacterial translocation and intraluminal toxins. Growing evidence suggests that the biological effects of radiation might be hormetic in nature. We investigated if exposure to low doses of ionizing radiation (LDR) can prevent colon hyposecretion due to subsequent larger doses. METHODS: Rats were exposed to LDR (0.05 Gy) 24 h prior to 6 Gy, high dose radiation (HDR). The cyclic adenosine monophosphate (cAMP)-mediated pathway was explored using forskolin (FSK) and the intracellular Ca2+-mediated pathway through cholinergic stimulation. Changes in the colonic epithelium at the ultrastructural level were also explored. RESULTS: Maximal short circuit current (Isc) response to carbachol was significantly reduced in the group exposed to 6 Gy HDR and this was completely prevented by prior exposure to LDR. Responses to both FSK and electrical field stimulation (EFS) were significantly reduced after HDR but they were not prevented by prior adaption of LDR. Hyposecretion was not prevented by the inducible nitric oxide synthase (iNOS) inhibitor L-N6-(l-iminoethyl)lysine (L-NIL) ruling out a role for iNOS-derived nitric oxide (NO) in the colonic hyposecretion associated with whole body radiation. Prior exposure to LDR diminished the deleterious effect of full HDR on the ultrastructure of colonic epithelium as colonocytes vacuolization, microvilli lost and separation between neighboring cells were less evident. CONCLUSIONS: Previous exposure to LDR can prevent intracellular Ca2+-mediated colonic hyposecretion associated with exposure to HDR but fails to modify cAMP-mediated hyposecretion. Morphological damage at the ultrastructural level is less evident after prior LDR.

New measurements of Cabibbo-suppressed decays of mesons with the CLEO-c detector.

Using of data collected with the CLEO-c detector, we report on first observations and measurements of Cabibbo-suppressed decays of D mesons in the following six decay modes: pi+ pi- pi0 pi0, pi+ pi+ pi- pi- pi0, pi+ pi0 pi0, pi+ pi+ pi- pi0, eta pi0, and omega pi+ pi-. Improved branching fraction measurements in eight other multipion decay modes are also presented. The measured D --> pi pi rates allow us to extract the ratio of isospin amplitudes A(DeltaI = (3/2) / A(DeltaI = (1/2)) = 0.420 +/- 0.014(stat) +/- 0.016(syst) and the strong phase shift of delta1 = (86.4 +/- 2.8 +/- 3.3) degrees, which is quite large and now more precisely determined.

Induction of LFA-1-dependent neutrophil rolling on ICAM-1 by engagement of E-selectin.

OBJECTIVE: To study rolling of mouse neutrophils on E-selectin and ICAM-1 in an ex vivo flow chamber system. METHODS: The authors developed a small autoperfused flow chamber (20 x 200-microm cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood. RESULTS: Neutrophils rolled on E-selectin alone, but were unable to interact with immobilized ICAM-1. When ICAM-1 was co-immobilized with E-selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E-selectin, and no enhancement of rolling was observed when P-selectin was immobilized with ICAM-1. The increased neutrophil rolling seen on E-selectin and ICAM-1 substrates required beta2 integrins. Treating mice with antibodies to the beta2 integrins LFA-1 and Mac-1 showed that LFA-1 was primarily responsible for mediating rolling on ICAM-1 in this model. Increased rolling on E-selectin and ICAM-1 was significantly reduced following administration of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. CONCLUSION: The data show that neutrophil rolling on E-selectin leads to partial activation of LFA-1, enabling LFA-1-dependent rolling on ICAM-1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation.

Absolute branching fraction measurements for D+ and D0 inclusive semileptonic decays.

We present measurements of the inclusive branching fractions for the decays D+-->Xe+ nu(e) and D0-->Xe+ nu(e), using 281 pb(-1) of data collected on the psi(3770) resonance with the CLEO-c detector. We find B(D0-->Xe+ nu(e)) = (6.46+/-0.17+/-0.13)% and B(D+-->Xe+ nu(e)) = (16.13+/-0.20+/-0.33)%. Using the known D meson lifetimes, we obtain the ratio Gamma(D+)sl/Gamma(D0)sl = 0.985+/-0.028+/-0.015, confirming isospin invariance at the level of 3%. The positron momentum spectra from D+ and D0 have consistent shapes.

Ion channels in interstitial cells of Cajal as targets for neurotransmitter action.

Interstitial cells of Cajal (ICC) are involved in generation of gut pacemaker activity, neurotransmission and stretch sensation. Pacemaker ICC exhibit spontaneous cyclic calcium oscillations that are in synchrony with its pacemaker activity. The spontaneous rhythmic inward currents in ICC that underlie gut pacemaker activity are linked to this calcium oscillation. It is probable that more than one type of channel contributes to the inward current with a high conductance chloride channel and a nonselective cation channel being the main candidates. The activation of these channels is linked to intracellular calcium cycling mechanism and involves inositol 1,4,5-trisphosphate (IP3)-mediated calcium release from the sarcoplasmic reticulum, and calcium uptake into mitochondria. This ion channel activity is modulated by signalling through neurotransmitter receptors, including the NK1 receptor. This finding and the presence of other neurotransmitter receptor mRNA transcripts indicates that ion channels in ICC are targets for neurotransmitter action. The ether-a-go-go-related (ERG) K channel is probably the most important K channel contributing to the resting membrane potential and excitability of the ICC. Many ion channels in ICC are regulated by second messenger systems which makes them highly susceptible to neurotransmitter modulation.

Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs.

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i)

Photoaffinity labeling of the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) with oligodeoxyribonucleotides.

The herpes simplex virus type-1 single-strand DNA-binding protein ICP8 is a 128-kDa zinc metalloprotein. In this communication we have shown that unsubstituted and bromodeoxyuridine-substituted oligonucleotides can be specifically crosslinked to ICP8 by UV irradiation. We have used this approach to show that the single-strand DNA-binding site of ICP8 resides within a 53.5-kDa tryptic polypeptide. This polypeptide initiates at alanine 368 and was estimated to extend through arginine 902. A polypeptide encompassing residues 368-902 synthesized in vitro exhibited single-strand DNA-binding activity. We conclude that the region encompassing residues 368-902 contains the single-strand DNA-binding site of ICP8. Moreover, photoaffinity labeling of ICP8 with oligonucleotides provides a means of specifically modifying its single-strand DNA-binding site, thereby facilitating future studies on the importance of its single-strand DNA-binding activity in its interaction with other DNA replication enzymes.

The N-terminal domain of MDM2 resembles calmodulin and its relatives.

It is shown here that the N-terminal domain of MDM2, which is not thought to bind calcium ions, otherwise bears a striking resemblance to a cluster of four EF-hand modules like those found in the calmodulin family. There are similarities in module arrangement, supersecondary structure and the main-chain to main-chain hydrogen-bonding pattern, especially in the vicinity of the short antiparallel beta-sheet, the two strands of which lie between the two E and F helices of tandem modules. Some conserved amino acid residues are identified that are associated with short side-chain to main-chain hydrogen-bonded motifs. Also, both types of domain bind a short, functionally important hydrophobic alpha-helix from another protein in a cavity between the two pairs of EF-hand, or EF-hand-like, modules.

Structural differences between valine-12 and aspartate-12 Ras proteins may modify carcinoma aggression.

Recent evidence associates the codon 12 valine-for-glycine (G12V) mutant Ki-Ras protein with higher stage and increased lethality of colorectal carcinomas, while the codon 12 aspartate-for-glycine (G12D) Ras mutation shows no such association. Several observations may be relevant to this phenomenon. First, GTPase activity of G12V Ras is one-quarter that of G12D Ras and one-tenth that of wild-type (WT) Ras. Second, binding of the GTP analogue GppNp to G12D Ras is 8-fold weaker than its binding to G12V or WT Ras and crystal structures indicate that electrostatic repulsion between the carboxylate group of the G12D Asp-12 side-chain and the gamma phosphate of the bound nucleotide may make GTP binding to G12D Ras weaker even than that of GppNp. It is proposed that this lowering of affinity for GTP allows G12D Ras an escape from the oncogenic GTP-bound state, whereas GTP tightly bound to G12V mutant Ras generates a more persistent, potentially oncogenic, signal. Structural comparisons also suggest that differences between the Switch I (effector) region of G12D and G12V Ras could modify interactions with downstream signalling molecules such as Raf-1, neurofibromin, and phosphatidylinositol 3-hydroxy-kinase. Other differences between the G12D and G12V mutant Ras proteins include a lower affinity of the GTPase activating protein GAP for G12V than for G12D or WT Ras; but, as both G12D and G12V Ras are refractory to GTPase activation by GAP binding, this may be less significant. These studies complement experimental data showing that such Ras mutations differ in their effects in vitro and in vivo and, with recent data indicating heterogeneity of ras mutation in colorectal carcinomas and other tumours, make it plausible that codon 12 Ras mutations differ in carcinogenic potential and prognostic significance.